The goal of this research is to understand the mechanisms by which a protein folds to a unique three-dimensional conformation. The system to be studied is a bacterial protein, the alpha subunit of tryptophan synthase. A large number of strains of Echerichia coli with missense mutations in the alpha gene region of the tryptophan operon have been isolated and the amino acid substitution in the alpha subunit identified. In vitro equilibrium and kinetic studies of the reversible unfolding transition of wild type and mutant proteins will monitor unfolding by changes in UV, circular dichroism, and NMR spectra. The results will be fit to simple models to allow a quantitative comparison of the effect of the amino acid replacement. From this comparison, it will be possible to learn which amino acids play a key role in the folding process. These conclusions will then be combined with structural information from x-ray studies and predictive schemes in order to understand the effects of the mutation at a molecular level. Knowledge of the involvement of these key amino acids in stabilizing secondary and tertiary structures in the native, folded conformation will permit an assessment of those features that are critical to folding. This information will be useful in predicting the tertiary structure from amino acid sequence and in understanding the molecular basis of inherited diseases.